There is controversy about the importance of glia-to-neuron communication in the adult brain, notwithstanding studies of gliotransmission 1, 2 and indications that astrocytes can modulate neuronal excitability and behavior in mammalian models 3, 4. For example, some have questioned the importance of glial Ca2+-dependent mechanisms in neuronal modulation 5-7. In addition, there is still limited knowledge of the mechanisms regulating gliotransmitter release, although certain studies suggest it involves vesicular exocytosis 2. In this application, we propose unbiased molecular genetic approaches to identify glial and neuronal factors essential for glia-to-neuron communication. We previously showed that elimination of a Drosophila glial-specific factor called Ebony genetically suppressed the hyperactivity phenotype of a Dopamine Transporter (DAT) mutant 8, indicating a role for glia-to-neuron communication in the regulation of activity level. In more recent published studies, we have demonstrated that adult Drosophila glial cells can physiologically modulate locomotor activity and circadian rhythmicity9. Our studies also demonstrate a critical role for Ca2+- dependent mechanisms and Drosophila astrocyte-like glia in the regulation of locomotor activity 9. In this R21 application, we propose studies to identify cell signaling mechanisms and intracellular pathways that are important for glia-to-neuron communication in adult animals. Drosophila is an excellent model for such studies as the glial classes of the adult brain have been well characterized 10-12 and one class has developmental, morphological and molecular similarities to mammalian astrocytes 11, 13, 14. Our studies will take advantage of multiple conditional perturbation methods that are cell type-specific and reversible to identify novel, conserved factors and intracellular pathways that mediate the glial modulation of neurons and behavior. Aim 1 will utilize Drosophila strains we recently developed and translational profiling methods to identify neuronal factors and molecular pathways that are modulated by glial signaling. Aim2 will use behavioral genetic strategies to define glial signaling mechanisms that are important for neuronal modulation. This proposal represents the first broad study of neuronal proteomic changes that occur as a consequence of glial signaling. It also represents the first genetic analysis of glial mechanisms that mediate the modulation of adult neurons. As such, the results will have a major impact on glial biology - they will define molecular pathways within neurons and glial cells that mediate glia-to-neuron signaling, and set the stage for studies of conserved factors in mammalian models to understand their functions in human health and neurological disease.